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Abstract:

High throughput system for in vivo screens on vertebrate larvae. The
system includes a source of vertebrate larvae in a liquid medium and
loading tube means for aspirating a larva. A detector assembly is
provided to differentiate passage of a larva from bubbles and/or debris.
An imaging means is provided for both confocal imaging and wide-field
fluorescence imaging of the larva. A laser is provided for optical
manipulation of the larva.

Claims:

1. High throughput system for in vivo screens comprising: a source of
specimen organisms in a liquid medium; an automatic means to load the
specimen organisms from the source into a tube; an automatic means to
position the specimen organism within the field-of-view of an imaging
apparatus; and a means to rotate the specimen organism.

2. The system of claim 1 wherein the specimen organism is a teleost.

3. The system of claim 1 wherein the source is a reservoir containing a
plurality of specimen organisms.

4. The system of claim 1 wherein the source is a multiwell plate
containing a plurality of specimen organisms.

5. The system of claim 3 wherein the source can be switched between the
specimen organisms reservoir and a liquid supply.

6. The system of claim 4 wherein a tube assembly is positioned on the
multiwell plate by a position stage.

7. The system of claim 6 wherein the tube assembly includes an injection
tube to inject fluid and an aspiration tube to aspirate a specimen
organism from the multiwell plate.

8. The system of claim 6 wherein the tube assembly and multiwell plate
can be sealed.

9. The system of claim 1 wherein the system includes an automatic
detector which has one or more light sources and one or more light
detectors arranged in transmission and/or reflection configurations.

10. The system of claim 9 wherein the automatic detector differentiates
air bubbles and/or debris from the specimen organisms.

11. The system of claim 1 wherein the tube includes a portion that is a
glass or quartz or transparent polymer capillary within the field-of-view
of the imaging apparatus.

12. The system of claim 1 wherein the imaging apparatus includes at least
one motor for rotating the tube within the field-of-view of the imaging
apparatus.

13. The system of claim 1 wherein the imaging apparatus simultaneously
images through two objective lenses.

15. The system of claim 1 wherein the imaging apparatus includes a laser
that generates a beam suitable for ablation and/or photoactivation and/or
photostimulation.

16. The system of claim 1 wherein the system is used to perform genetic
screens.

17. The system of claim 1 wherein the system is used to perform chemical
screens.

18. Method for high-throughput in vivo screens comprising: providing a
source of specimen organisms in a liquid medium; automatically loading
the specimen organisms from the source; automatically positioning the
specimen organisms within the field-of-view of an imaging apparatus; and
rotating the specimen organisms.

19. The method of claim 18 further including sorting the specimen
organisms into multiple containers.

20. The method of claim 18 including running the specimen organisms
through the system multiple times.

21. The method of claim 18 further including exposing the specimen
organism to a compound to image acute effects.

22. The method of claim 18 for high throughput in vivo cell counting.

23. The method of claim 18 for measuring morphological changes in vivo.

24. The method of claim 18 for measuring cellular morphology.

25. The method of claim 18 for measuring cellular migration.

26. The method of claim 18 for measuring Ca2+ signals.

27. The method of claim 18 used for optical manipulation at cellular
resolution using laser microablation.

28. The method of claim 18 used for photostimulation of ion-channels.

29. The method of claim 18 used for photoactivation or photoinactivation
of chemicals and proteins.

30. The method of claim 18 used for regeneration screens of chemicals
that enhance proliferation of progenitor cells.

31. The method of claim 18 used for tumor screens for inhibitory
chemicals targeting tumorigenic cell proliferation in vivo.

32. The method of claim 18 used for screens for protective chemicals that
reduce or halt cell death in the degenerative disease models.

33. The method of claim 18 used for toxicity screens.

34. The method of claim 18 used for genetic and chemical screens on
development and organogenesis.

35. The method of claim 18 for screens on neuronal morphology such as
neurite branching, growth, connectivity, and spine density.

36. The method of claim 18 used for screens on stem cell migration.

37. The method of claim 18 used for screens on cardiovascular function
and health.

38. The method of claim 27 used for screens enhancing regeneration of
specific tissues after precise injury.

39. The method of claim 27 used for screens compensating function after
ablation of specific cells.

40. The method of claim 18 used for screens on cell lineages and
differentiation.

41. The system of claim 2 wherein the teleost is zebrafish.

42. The system of claim 1 wherein the specimen organism is transgenic.

43. An automated system for in vivo screens on vertebrate larvae
comprising: a source of vertebrate larvae in a liquid medium; means to
aspirate a larva into a tube; a detection means to differentiate passage
of a larva from bubbles and/or debris; a means to rotate the organism;
and a means for imaging the organism.

Description:

BACKGROUND OF THE INVENTION

[0001] This invention relates to a high throughput screening platform and
more particularly to a high throughput platform capable of in vivo
genetic and chemical screens on specimen organisms such as zebrafish
larvae and other teleosts.

[0002] Small animal models such as zebrafish (Danio Rerio) facilitate the
study of complex processes on a large scale that cannot be replicated in
vitro such as: organ development; neural degeneration and regeneration;
stem cell proliferation and migration; cardiovascular, immune, endocrine,
and nervous system functions; infectious disease progression;
pathogenesis; cancer progression; and tissue specificity and toxicity of
drugs. Several desirable attributes of zebrafish have fueled its
popularity, including the animal's small size, optical transparency,
aquatic habitat, and simplicity of culture. Zebrafish models of several
human diseases have been developed1-11. Superscript numbers refer to
the references included herewith. The contents of all these references
are incorporated herein by reference. Lead compounds discovered by
screening chemical compound libraries for efficacy in zebrafish disease
models have been useful for pharmaceutical development because of the
high level of conservation of drug activity between mammals and
zebrafish12,13. The availability of large numbers of mutant strains
and genetic manipulations such as gene overexpression, knockdown, and
silencing make zebrafish a powerful model for genetic studies and for
identification of the cellular targets of new compounds1,14,15. The
significant advantages of zebrafish have fueled exponential growth of its
use in experimental investigations over the last two decades1.

[0003] Several companies and academic labs are conducting genetic and
compound screens on zebrafish larvae incubated in 96-well
plates1,2,4,12. Because handling of zebrafish has been largely
manual, typical high-content zebrafish screens are limited to only a few
thousand compounds per week. Subcellular resolution assays require
optical access to a specific region of the specimen for imaging or
manipulation. Clear access is often impeded by intervening organs such as
eyes and heart. Yolk and some organs exhibit significant
autofluorescence. In addition, skin pigmentations can block the region of
interest. Visualization of most of the regions requires orienting the
zebrafish appropriately. However, current specimen orientation methods
require embedding the sample in viscous media such as agar and/or
manually rotating the fish with forceps. These processes are too slow and
unreliable for high-throughput screens. In addition, specimens cannot be
rapidly re-oriented once they are fixed, thus impeding visualization of
organs from multiple angles. Examples of assays that require sample
orientation and subcellular resolution imaging include in vivo monitoring
of early tumor growth, neuronal degeneration, neurite regeneration, and
stem cell proliferation and migration in tissues comprising the brain,
eyes, heart, pancreas, kidneys, and liver.

SUMMARY OF THE INVENTION

[0004] In one aspect, the high throughput system of the invention for in
vivo screens includes a source of specimen organisms in a liquid medium.
Suitable specimen organisms include teleosts or other aquatic animals and
embryos. A preferred teleost is zebrafish larvae. An automatic means is
provided to load the specimen organisms from the source into a tube. An
automatic detector detects passage of the specimen organisms through the
tube. Also provided is an automatic means to position the specimen
organism in the tube within the field-of-view of an imaging apparatus.

[0005] In a preferred embodiment, the larvae are zebrafish. It is
preferred that the source of larvae be a reservoir or a multiwell plate.
The reservoir may include a fish reservoir and a liquid reservoir. It is
preferred that the multiwell plate be a 96 well plate and that it sits on
an x-y position stage. In yet another preferred embodiment, the detector
assembly includes two light emitting diodes and one high-speed photodiode
arranged in transmission and reflection configurations. In an embodiment,
the imaging means includes a pair of stepper motors for rotating a
capillary tube to reorient a larva. The capillary tube may preferably be
glass or Teflon.

[0006] In yet another embodiment, the imaging means includes a 20 power
water-immersion objective lens for confocal imaging. The imaging means
also includes an inverted 10 power objective lens for brightfield
imaging. It is preferred that the imaging means include a cooled
electron-multiplying CCD camera. It is preferred that the laser be a
femtosecond laser. The femtosecond second laser is selected to generate a
beam suitable for microsurgery or ablation and/or photoactivation. The
system disclosed herein may be used to perform genetic or chemical
screens.

BRIEF DESCRIPTION OF THE DRAWING

[0007] FIGS. 1a and 1b are the schematic flow diagrams for chemical and
genetic screens according to an embodiment of the invention.

[0011] To facilitate a dramatic improvement in the throughput and
complexity of zebrafish screens, we developed a platform for rapid
manipulation of zebrafish larvae. Zebrafish larvae will be used herein to
describe the invention. It is to be recognized that other teleosts and
aquatic animals and embryos may be used and it is intended that other
such specimen organisms be included within the term zebrafish larvae. The
automated system allows both genetic and chemical screens, as shown in
FIG. 1. For genetic screens, mutagenized animals are loaded from a
reservoir to the imaging platform. After imaging, the mutants are sorted
into multilevel plates by phenotype. For chemical screens, animals are
loaded from a reservoir to the imaging platform which can be used either
to count the number of animals and/or to perform optical manipulations
such as laser microsurgery. The animals are then dispensed into multiwell
plates containing chemicals to be tested. After incubation within
chemicals, the animals are loaded back into the imaging platform to check
phenotypes.

[0012] Examples of genetic and chemical screens that can be performed with
the present invention include a chemical screen for organ regeneration
searching for promoters of progenitor cells proliferation. This assay can
be performed in multiple organs such as the liver, the kidneys, the
pancreas, skeletal muscle, hair follicles and the central and peripheral
nervous system. Another example is screens for inhibitory chemicals
targeting tumorigenic cell proliferations in vivo. Yet another example is
screens for protective chemicals that significantly reduce or inhibit
cell hypotrophy or death in degenerative disease models such as
Parkinson's Disease, Alzheimer's or Huntington's Disease. Screens to
assess biological toxicity of drugs in multiple organs, such as in the
liver, kidneys, pancreas, heart, hair cells and neurons in the central
and peripherial nervous systems are contemplated. Genetic and chemical
screens to study the differentiation and proliferation of different cell
lineages and organogenesis by photoactivation of fluorescent reporter
proteins as lineage tracers may also be preformed using the system of the
invention disclosed herein.

[0013] The capability to work with standard multiwell plates is essential
for handling and incubating large populations of zebrafish larvae, so the
system was designed to automatically load specimens from and dispense
them back into individual wells. The system also has the ability to load
animals from reservoirs. After automatic loading, animals are positioned
within the field-of-view (FOV) of an optical imaging and manipulation
subsystem with high-speed confocal imaging and optical manipulation
capabilities. Specimens can be repositioned and rotated on the fly,
eliminating the need for manual handling and invasive chemical methods to
suppress skin pigmentation such as phenylthiourea (PTU) treatment16.
Furthermore, active control of specimen orientation permits the use of
high numerical aperture (NA) objective lenses that require short working
distances. High NA objectives collect more light, reduce background
fluorescence, and diminish scattering and absorption by intervening
tissue. Using our system, high-content subcellular-resolution screens are
possible.

[0014] With reference now to FIG. 2a a liquid medium reservoir 10 and a
fish reservoir 12 are controlled with fluidic valves 14 and 16. A bubble
mixer 18 injects bubbles into the fish reservoir 12 to provide agitation.
A syringe pump (not shown) allows fish larvae to be aspirated into a tube
20. After one animal enters the tube, the two computer-controlled valves
14 and 16 automatically switch the fluid source from the larvae reservoir
12 to a separate reservoir 10 containing fish medium to prevent loading
of multiple fish.

[0015] In an alternative embodiment as shown in FIG. 2b, larvae are
included in a multiwell plate 21 supported on an x-y position stage. The
2-axis stage positions a single well beneath a pneumatic piston that
lowers the endpoints of two silicon tubes (loading and supply tubes with
diameters of 1.0 and 0.2 mm, respectively) into the volume of the
selected well. A silicone rubber block 22 seals a well surface to
facilitate depressurization. The larva is aspirated through a loading
tube while a supply tube simultaneously replenishes the fluid removed.

[0016] A zebrafish larva from either the fish reservoir 12 or from a well
in the multiwell plate 21 passes through a detector section of the system
as shown in FIG. 2c. The detector system automatically discerns the entry
of larvae into the tube 20 from either the wells 21 or the reservoir 12
and includes two light emitting diodes 24 and 26 and a photodetector 28
positioned around the tube 20. The light emitting diode 26 is arranged so
that its light is transmitted through a larva and received by the
photodetector 28. The light emitting diode 24 is offset so that light
reaching the photodetector 28 has been scattered. By simultaneously
monitoring both the transmission and scattering signals, the system of
the invention differentiates the passage of a larva from air bubbles and
debris. To ensure recognition of the rapidly moving animals, the detector
operates at a preferred sampling rate of 2 kHz.

[0017] A larva moves from the detector section shown in FIG. 2c into an
image system shown in FIG. 2d. As shown in FIG. 2d, a larva 30 is located
within the FOV in a Teflon capillary tube 32 having an inner diameter of
approximately 800 μm. The capillary 32 is matched to the refractive
index of water to prevent image distortion. The capillary tube 32 may be
glass.

[0018] As shown in FIG. 2d, the optical imaging system includes two
microscopes: one upright and the other inverted. The upright lens 34 is a
20× (NA=1.0) water-immersion objective lens for confocal imaging
and an inverted 10× lens 36 is an air objective lens for
brightfield imaging. A high-speed spinning-disk confocal head with a
cooled electron-multiplying EM CCD camera is connected to the
microscope's upright port for video frame rate confocal fluorescence
imaging. The electron-multiplying CCD camera is element 38. A second CCD
camera 40 is connected to an inverted port for detection and positioning
of larvae. A femtosecond laser 42 provides a beam used for microsurgery
or ablation and photoactivation or photostimulation and is directed to
the upper beam path by a dichroic filter and focused on the sample
through the 20× objective 34. Two stepper motors 44 and 46 are
precisely controlled to rotate in opposite directions so as not to twist
the capillary 32 thereby providing for accurate location of the larva 30.

[0019] A computer-controlled syringe pump (not shown) automatically
controls coarse positioning of a larva inside the capillary tube 32
within the field of view of the optical subsystem of FIG. 2d. The
capillary 32 is matched to the refractive index of water to prevent image
distortion. Using images from a fast EMCCD camera, an automated image
processing algorithm detects the entrance of a larva into the field of
view and subsequently stops the syringe pump. After coarse positioning of
the larva with the syringe pump, a 3-axis position stage automatically
moves the capillary assembly to position the animal's head at the center
of the camera's field of view via automated image processing as is known
in the art.

[0020] The animals are oriented on-the-fly by a pair of stepper motors 44
and 46 that rotate the capillary 32. The motors 44 and 46 are mounted on
a support structure with their shafts facing one another. The motors are
driven by a microstep controller connected to a computer (not shown)
which allows 0.2 degree steps at an angular velocity of up to 180 degrees
per second. The entire motor and capillary assembly, plus a water filled
imaging chamber in which the top objective is immersed, is mounted on the
3-axis motorized position stage. Thus, larvae can be arbitrarily
positioned and oriented within the microscope's field of view with
submicron accuracy. The imaging system allows simultaneous wide-field
fluorescence imaging and high speed, high-resolution, spinning disk
confocal microscopy. The imaging system may have two-photon imaging
capability.

[0021] FIG. 3a shows an example set of confocal images of one specimen
oriented at several angles to visualize the midline crossing of the
Mauthner neuron axons17. The midline crossing is only visible when
observed from the hindbrain (0° in the figure). At less favorable
orientations, the structure is obscured. Images are shown additionally at
15° and 45°. The scale bar is 150 μm.

[0022] The system also incorporates a subcellular-precision, femtosecond
laser stimulation capability. Combined with the precise automatic
positioning and orientation capabilities of the system, the femtosecond
laser 42 enables optical manipulations such as localized activation of
fluorescent reporters and ion channels, uncaging of compounds, and laser
microsurgery. FIG. 3b shows an example of how the system would be used to
study neuronal regeneration following injury by laser microsurgery.
Zebrafish have significant regenerative capacity, which makes them a
powerful model for investigating regenerative mechanisms as well as for
screening chemicals that enhance regeneration. Existing assays for
injuring zebrafish using scalpels are too slow and invasive for reliable,
large-scale studies of regeneration18. In the image, the
lateral-neuron axon fiber bundle projecting along the trunk of a larva is
visible when the larva is laterally oriented. Precise laser axotomy is
achieved by focusing near infrared (NIR) femtosecond laser pulses (white
arrow in FIG. 3b) as we previously demonstrated for study of neuronal
regeneration in C. elegans13. At NIR wavelengths, the tissue is
highly transparent; however, nonlinear absorption of photons at the focus
of the ultra-short pulses allows micron-precision axon ablation without
damaging surrounding tissue. The surgery is done semi-automatically in
our system for highest throughput. The user selects a cell body by
clicking on a graphical user interface. An algorithm estimates the
distance from the cell body to the point of axotomy along the axon. The
position stage automatically moves the axonal region to be axotomized to
the focal spot of the laser 42. Femtosecond laser 42 pulses are then
automatically delivered by opening an electro-optical laser shutter to
perform the microsurgery. FIG. 3b also shows the regrown axonal fibers at
18 and 24 hours post-axotomy. The axon fiber is cut 850 μm distance
from the soma using ultrashort laser pulses with 780 nm wavelength, 100
fs duration, 15 nj pulse energy, 80 MHz repetition rate and 10 ms long
pulse train focused by a 20× NA=1.0 objective lens. Scale bar is 75
μm.

[0023] After imaging and manipulation, larvae are dispensed into multiwell
plates for further incubation by executing the loading process in
reverse. The dispensed solution volume is hydraulically controlled to a
precision greater than 10 μL. A complete cycle consisting of loading,
positioning, rotating, sub-cellular resolution confocal imaging, and
dispensing an animal takes less than 16 seconds (FIG. 4a). Performed
manually, similar assays require about 10 minutes, and the failure rate
is much higher.

[0024] Zebrafish larvae are delicate; their yolks are particularly
susceptible to indelicate handling.

[0025] We tested alternative methodologies and flow rates to develop a
minimally injurious system. The most significant damage to the larvae
occurs during its entry from the reservoir 12 or multiwell plate 21 into
the loading tube 20 at high aspiration rates. Since high aspiration rates
are necessary for acceptable throughput, the flow is started at a low
initial rate before an animal enters the loading tube to reduce the
chance of injury, and the rate is then increased at a constant
acceleration of 42 μl/s2 until a larva is detected by the
photodetector (FIG. 2c). The maximum flow rate is limited to 330 μl/s.
At this maximum flow rate, no injury to larvae occurs if the larva is
within the tubing. Once a larva is detected inside the capillary 32, the
control software automatically decreases the aspiration rate to 83
μl/s to allow automated recognition of the larva by the camera 38.
FIG. 4b shows the results for 450 larvae screened by the system at three
different initial aspiration rates. After 36 hours, larval health was
assessed by visual confirmation of normal heartbeat, morphology, and
reflex response to touch and light stimuli. At the highest initial flow
rate (330 μl/s), 2.0% of the animals exhibited a morphological
abnormality. The abnormality criteria included lordosis, kyphosis,
scoliosis and craniofacial abnormalities19. Post manipulation
developmental delay was measured by monitoring the time of appearance of
the swimming bladder. As shown in FIG. 4c, there was no significant
(P=0.94) difference between development of larvae that were manipulated
by the system and control animals, even at the highest flow rates.
Furthermore, when operating at a slightly slower loading rate (increasing
the cycle time approximately by 1 second), the system had no effect on
survivability.

[0026] We have demonstrated a high-throughput platform for manipulating a
specimen organism with cellular-resolution imaging and surgery
capabilities, enabling complex in vivo high-throughput assays such as
neuronal regeneration. The platform can be used for both forward and
reverse genetic screens, as well as for chemical screens. The platform
automatically loads and dispenses animals from multiwell plates, which is
crucial for large-scale incubation of zebrafish. It is capable of
orienting animals on-the-fly with sub-degree precision and therefore
allowing visualization and manipulation of superficial and deep
structures that are otherwise inaccessible in existing high-throughput
screens. The entire process of loading, positioning, orienting, imaging,
laser micromanipulation, and dispensing of animals takes place within 18
seconds, an improvement of about two orders of magnitude over existing
methods. Screening hundreds of animals demonstrated that our system works
noninvasively and reliably in the presence of artifacts such as air
bubbles and debris in the medium. Thus, our platform can significantly
accelerate the throughput of sophisticated assays on vertebrates.

[0027] It is recognized that modifications and variations of the invention
will be apparent to those of ordinary skill in the art, and it is
intended that all such modifications and variations be included within
the scope of the appended claims.